Instrument for Monitoring Polymerase Chain Reaction of DNA

ABSTRACT

An optical instrument monitors PCR replication of DNA in a reaction apparatus having a temperature cycled block with vials of reaction ingredients including dye that fluoresces in presence of double-stranded DNA. A beam splitter passes an excitation beam to the vials to fluoresce the dye. An emission beam from the dye is passed by the beam splitter to a CCD detector from which a processor computes DNA concentration. A reference strip with a plurality of reference emitters emit reference beams of different intensity, from which the processor selects an optimum emitter for compensating for drift. Exposure time is automatically adjusted for keeping within optimum dynamic ranges of the CCD and processor. A module of the beam splitter and associated optical filters is associated with selected dye, and is replaceable for different dyes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/333,482, filed Jan. 17, 2006, which in-turn is acontinuation of U.S. patent application Ser. No. 10/216,620, filed Aug.9, 2002, now U.S. Pat. No. 7,008,789 B2, which in-turn is a continuationof U.S. patent application Ser. No. 09/700,536, filed Nov. 29, 2001, nowU.S. Pat. No. 6,818,437 B1, which in-turn is a National PhaseApplication Under 35 U.S.C. §371 of PCT International Application No.PCT/US99/11088, filed on May 17, 1999, which claims priority benefitsfrom U.S. Provisional Patent Application No. 60/085,765, filed May 16,1998, and from U.S. Provisional Patent Application No. 60/092,784, filedJul. 14, 1998, all of which are hereby incorporated herein in theirentireties by reference.

This invention relates to biochemical analyses, and particularly toquantitative monitoring of DNA during a polymerase chain reaction (PCR)process.

BACKGROUND

Polymerase chain reaction (PCR) is a process for amplifying ormultiplying quantities of double-stranded deoxyribonucleic acid (DNA).In a PCR apparatus, a thermal cycler block has one or more wells forholding vials containing a suspension of ingredients for a reaction toproduce more DNA starting with “seed” samples of the DNA. The startingingredients in an aqueous suspension, in addition to the a seed sample,include selected DNA primer strands, DNA elements, enzymes and otherchemicals. The temperature of the block is cycled between a lowertemperature extension phase of the PCR reaction at about 60° C., whichis the phase where all of the DNA strands have recombined into doublestrands, and a high temperature denaturing phase at about 95° C., duringwhich the DNA is denatured or split into single strands. Such atemperature program essentially doubles the DNA in each cycle, thusproviding a method for replicating significant amounts of the DNA from asmall starting quantity. The PCR process is taught, for example, in U.S.Pat. No. 4,683,202.

Quantitative measurements have been made on the DNA production duringthe PCR process, to provide measures of the starting amount and theamount produced. Measurements and computation techniques are taught inU.S. Pat. No. 5,766,889 (Atwood), as well as in an article “Kinetic PCRAnalysis: Real-time Monitoring of DNA Amplification Reactions” by RusselHiguchi, et al., Bio/Technology vol. 11, pp. 1026-1030 (September 1993),and an article “Product Differentiation by Analysis of DNA MeltingCurves during the Polymerase Chain Reaction” by Kirk M. Ririe, et al.,Analytical Biochemistry vol. 245, pp. 154-160 (1997).

Prior measuring techniques have utilized microvolume fluorometers(spectrofluorometers) and a simple arrangement of a video camera withillumination lamps. Such apparatus utilize dyes that fluoresce in thepresence of double-stranded DNA. These techniques and instruments arenot particularly adapted to PCR apparatus for routine monitoring of thereaction. There also is a need for greater precision during themonitoring and measurements. Previous instruments that allow real timeacquisition and analysis of PCR data have been very basic deviceswithout the required dynamic range, do not have built-in calibrationmeans, do not allow operation with sample well caps, or are veryexpensive.

An object of the present invention is to provide a novel opticalinstrument for quantitative monitoring of DNA replication in a PCRapparatus. Other objects are to provide such an instrument with improveddynamic range, automatic selection of exposure time to extend dynamicrange, automatic adjustment for drift, simplified operation, relativelylow cost, and easy changing of optics to accommodate differentfluorescent dyes.

SUMMARY

The foregoing and other objects are achieved, at least in part, by anoptical instrument as described herein for monitoring polymerase chainreaction replication of DNA. The replication is in a reaction apparatusthat includes a thermal cycler block for holding at least one vialcontaining a suspension of ingredients for the reaction. The ingredientsinclude a fluorescent dye that fluoresces proportionately in presence ofDNA.

The instrument includes a light source, means for directing light beams,a light detector, and means for processing data signals. The lightsource emits a source beam having at least a primary excitationfrequency that causes the dye to fluoresce at an emission frequency. Afirst means is disposed to be receptive of the source beam to effect anexcitation beam having the excitation frequency. A primary focusingmeans is disposed to focus the excitation beam into each suspension suchthat the primary dye emits an emission beam having the emissionfrequency and an intensity representative of concentration of DNA ineach suspension. The focusing means is receptive of and passes theemission beam. A second means is disposed to be receptive of theemission beam from the focusing means so as to further pass the emissionbeam at the emission frequency to another focusing means that focusesthe emission beam onto a detector. The detector generates primary datasignals representative of the emission beam and thereby a correspondingconcentration of DNA in each vial. A processor is receptive of theprimary data signals for computing and displaying the concentration ofDNA.

In a preferred embodiment, the first means and the second means togethercomprise a beam splitter that is receptive of the source beam to effectthe excitation beam, and receptive of the emission beam to pass theemission beam to the detector. The block is configured to hold aplurality of vials, and the focusing means comprises a correspondingplurality of vial lenses each disposed over a vial such that theemission beam comprises individual beams each associated with a vial.The focusing means may further comprise a field lens such as a Fresnellens disposed cooperatively with the vial lenses to effect focusing ofthe excitation beam into each suspension, and to pass the individualbeams to the second means (beam splitter). The detector preferablycomprises an array of photoreceptors receptive of the individual beamsto generate corresponding data signals such that the processing meanscomputes concentration of DNA in each vial.

The instrument should also include an excitation filter between thelight source and the beam splitter, and an emission filter between thebeam splitter and the detector. The splitter and filters are associatedwith a selected primary dye in the suspension. In a further embodiment,a filter module contains the splitter and filters, and the module isremovable from the housing for replacement with another moduleassociated with another selected primary dye.

For a reference, a fluorescent reference member emits reference light inresponse to the excitation beam. The reference is disposed to bereceptive of a portion of the excitation beam from the first means. Aportion of the reference light is passed by the second means as areference beam to the detector, so as to generate reference signals forutilization in the computing of the concentration of DNA. Preferably thereference member comprises a plurality of reference emitters, eachemitting a reference beam of different intensity response to theexcitation beam, to allow selection by the processor of a reference sethaving the highest data signals that are less than a predeterminedmaximum that is less than the saturation limit.

The detector is operatively connected to the processing means for thedetector to integrate emission beam input over a preselected exposuretime for generating each set of data signals, and the processing meansor the detector or a combination thereof have a saturation limit for thedata signals. In a further aspect of the invention, the processing meanscomprises adjustment means for automatically effecting adjustments inexposure time to maintain the primary data within a predeterminedoperating range for maintaining corresponding data signals less than thesaturation limit, and means for correcting the primary data inproportion to the adjustments in exposure time. Preferably, theprocessor computes photoreceptor data from the data signals for eachphotoreceptor, and the adjustment means ascertains highest photoreceptordata, determines whether the highest photoreceptor data are less than,within or higher than the predetermined operating range and, based onsuch determination, the exposure time is increased, retained or reducedso as to effect a subsequent exposure time for maintaining subsequentphotoreceptor data within the predetermined operating range.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an optical train for an optical instrumentaccording to the invention, associated with a polymerase chain reaction(PCR) reaction apparatus.

FIG. 2 is a perspective of the instrument of FIG. 1 with a side panelremoved.

FIG. 3 is an exploded perspective of a module shown in FIG. 2.

FIG. 4 is a perspective of a reference member in the optical train ofFIG. 1.

FIG. 5 is a flow chart for computing DNA concentration from dataobtained with the instrument of FIG. 1.

FIG. 6 is a flow chart for determining exposure time for dataacquisition in operation of the instrument of FIG. 1 and forcomputations in the flow chart of FIG. 5.

FIG. 7 is a graph of extension phase data of fluorescence vs. cyclesfrom operation of the instrument of FIG. 1 with a PCR apparatus.

FIG. 8 is a flow chart for computing secondary data for computations inthe flow chart of FIG. 5.

FIG. 9 is a flow chart for computing ratios between the plurality ofreference emitter segments of the reference member of FIG. 4.

DETAILED DESCRIPTION

An optical instrument A of the invention is utilized with orincorporated into a reaction apparatus B that replicates (“amplifies”)selected portions of DNA by polymerase chain reaction (“PCR”). Thereaction apparatus is conventional and should function withoutinterference from the instrument which monitors the amount of DNA inreal time during replication. Suitable reaction apparatus are describedin U.S. Pat. Nos. 5,475,610 and 5,656,493.

The reaction apparatus (FIG. 1) is conventional and has two maincomponents, namely a thermal cycler block 1 with wells 1 a for holdingat least one vial 1 b containing a suspension of ingredients for thereaction, and a thermal cycle controller 1 c for cycling the temperatureof the block through a specified temperature program. The startingingredients of the aqueous suspension of sample materials include a“seed” sample of DNA, selected DNA primer strands, DNA elements, enzymesand other chemicals. The block, typically aluminum, is heated and cooledin a prescribed cycle by electrical means, liquid or air coolant, or acombination of these, or other means to achieve the cycling. Thesuspensions in the vials are thereby cycled between two temperaturephases so as to effect the polymerase chain reaction. These phases are alower temperature extension phase of the PCR reaction at about 60° C.,which is the phase where all of the DNA strands have recombined intodouble strands, and a high temperature denaturing phase at about 95° C.,during which the DNA is denatured or split into single strands.

For the present purpose the sample also contains a fluorescent dye thatfluoresces proportionately and more strongly in the presence of doublestranded DNA to which the dye binds, for example SYBR Green dye(available from Molecular Probes, Inc., Eugene, Oreg.) that fluorescesin the presence of double stranded DNA. Another type of fluorescent dyelabeled “probes”, which are DNA-like structures with complimentarysequences to selected DNA strand portions, may also be used. Other dyesthat have similar characteristics may be utilized. As used herein and inthe claims, the term “marker dye” refers to the type that binds todouble stranded DNA, or to the probe type, or to any other type of dyethat attaches to DNA so as to fluoresce in proportion to the quantity ofDNA. Samples may also contain an additional, passive dye (independent ofthe DNA) to serve as a reference as described below. Under incidence oflight having a correct excitation frequency, generally a dye fluorescesto emit light at an emission frequency that is lower than that of theexcitation light.

The vials typically are formed conically in a plastic unitary traycontaining a plurality of vials, for example 96 in an array of 12 by 8.The tray preferably is removable from the block for preparations. Aplastic unitary cover with caps 1 d for the vials may rest or attachover the vials to prevent contamination and evaporation loss. Othermeans may be used for this function, such as oil on the sample surface,in which case caps are not needed. If used, the caps are transparent tolight utilized in the instrument, and may be convex facing upwardly.

The monitoring instrument is mounted over the block containing thevials. The instrument is removable or swings away for access to thevials. In the bottom of the instrument, a platen 2 rests over the vialcaps or, if none, directly over the vials. The platen, advantageouslyaluminum, has an array of holes 2 a therethrough aligned with the vials,each hole having a diameter about the same as the vial top diameter. Ifthere are caps, the platen should have its temperature maintained by afilm heater or other means for heating the platen sufficiently toprevent condensation under the caps without interfering with DNAreplication in the vials, for example holding the platen at slightlyhigher temperature than the highest sample temperature that the thermalcycler reaches.

Above each of the vials is a lens 2 b positioned for its focal point tobe approximately centered in the suspension in the vial. Above theselenses is a field lens 3 to provide a telecentric optical system.Advantageously the field lens is an aspherically corrected Fresnel lensfor minimal distortion. A neutral density pattern (not shown) to correctnonuniformities in illumination and imaging may be mounted on or inproximity to the field lens, for example to attenuate light in thecenter of the image field. A folding optical mirror is optionallymounted at 45° for convenient packaging. This may be omitted, or othersuch folding optics may be used. Also the field lens, and/or the viallenses, each may be comprised of two or more lenses that effect therequired focusing, the word “lens” herein including such multiplicities.

A light source 11 for a source beam 20 of light is provided, for examplea 100 watt halogen lamp. Preferably this is mounted at a focal distanceof an ellipsoid reflector 11 a which produces a relatively uniformpattern over the desired area. Also, advantageously, the reflectorshould be dichroic, i.e. substantially reflecting visible light andtransmitting infrared light, to restrict infrared from the other opticalcomponents and from overheating the instrument. This is further aided bya heat reflecting mirror 13 in the optical path. A mechanical orelectronic shutter 12 allows blockage of the light source for obtainingdark data. The type of light source is not critical, and other types maybe used such as a projection lamp or a laser, with appropriate opticalelements.

A beam splitter 6 is disposed to receive the source beam 20. In thepresent embodiment this is a dichroic reflector such that, positioned at45°, it reflects light having an excitation frequency that causes themarker dye to fluoresce at an emission frequency, and passes lighthaving the emission frequency. Such a conventional optical devicetypically utilizes optical interference layers to provide the specificfrequency response.

The beam splitter is positioned to reflect the source beam to thefolding mirror. The source beam is reflected from the splitter as aexcitation beam 22 having substantially the excitation frequency. Theexcitation beam is focused by the field lens 3 and then as separatedbeams 24 by the vial (well) lenses 2 b into the center of the vials. Themarker dye is thereby caused to emit light at the emission frequency.This light is passed upwardly as an emission beam in the form ofindividual beams 26 that are reflected from the folding mirror 5 to thebeam splitter 6 which passes the emission beam through to a detector 10.

Together the vial lenses 2 b and the field lens 3 constitute a primaryfocusing means for is focusing both the excitation beam and the emissionbeam. In an alternative aspect, the field lens may be omitted so thatthe focusing means consists only of the vial lenses 2 b. Alternatively,the vial lenses may be omitted so that the focusing means consists onlyof an objective lens in the field lens position to focus the individualemission beams on the detector.

Also, alternatively, the beam splitter 6 may pass the source beam as anexcitation beam and reflect the emission beam, with appropriaterearrangement of the lamp and the detector. Moreover, other angles than45° could be used if more suitable for the beam splitter, such as a moreperpendicular reflection and pass through. More broadly, the beamsplitter splits the optical paths for the excitation beam and theemission beam, and other variations that achieve this may be suitable.It is desirable to minimize source light reaching the detector, whichthe dichroic device helps achieve. A non-dichroic beam splitter may beused but would be less efficient as significant source light may reachthe detector, or may be reflected or transmitted in the wrong directionand lost.

To further filter the source light, an excitation filter 7 is disposedbetween the light source 11 and the beam splitter 6. This passes lighthaving the excitation frequency and substantially blocks light havingthe emission frequency. Similarly, an emission filter 8 is disposedbetween the beam splitter and the detector, in this case between thesplitter and a detector lens 9 in front of the detector. This filterpasses light having the emission frequency and substantially blockslight having the excitation frequency. Although a detector lens ispreferred, a focusing reflector may be substituted for the detectorlens. Such an emission focusing means (detector lens or reflector) maybe located after (as shown) or before the beam splitter and on eitherside of the emission filter, and alternatively may be integrated intothe primary focusing means. For example, the field lens may be anobjective lens that focuses the emission beam onto the detector.

Suitable filters are conventional optical bandpass filters utilizingoptical interference films, each having a bandpass at a frequencyoptimum either for excitation of the fluorescent dye or its emission.Each filter should have very high attenuation for the other(non-bandpass) frequency, in order to prevent “ghost” images fromreflected and stray light. For SYBR Green dye, for example, theexcitation filter bandpass should center around 485 nm wavelength, andthe emission filter bandpass should center around 555 nm. The beamsplitter should transition from reflection to transmission between thesetwo, e.g. about 510 nm, so that light less than this wavelength isreflected and higher wavelength light is passed through.

More broadly, the excitation filter and the beam splitter togetherconstitute a first means disposed to be receptive of the source beam toeffect an excitation beam having the excitation frequency, and theemission filter and the beam splitter together constitute a second meansdisposed to be receptive of the emission beam from the focusing means soas to pass the emission beam at the emission frequency to the detector.Also, as mentioned above, the beam splitter alternatively may pass thesource beam as an excitation beam and reflect the emission beam to thedetector. In another aspect, the filters may be omitted, and the firstmeans is represented by the beam splitter effecting the exitation beamfrom the source beam, and the second means is represented by the beamsplitter passing the emission beam to the detector.

In another arrangement, the beam splitter may be omitted, and the firstmeans may constitute an excitation filter for the excitation frequency,the second means may constitute an emission filter for the emissionfrequency, with the light source and the detector being side by side sothat the excitation and emission beams are on slightly different opticalpaths angularly. The source and detector need not actually be side byside with one or more folding mirrors. Thus any such arrangement forachieving the effects described herein should be deemed equivalent.However, use of the beam splitter is preferred so that the excitationand emission beams through the field lens will have the same opticalpath.

Advantageously the beam splitter 6, the excitation filter 7 and theemission filter 8 are affixed in a module 30 (FIG. 2) that is associatedwith a selected primary dye for the suspension. The module is removablefrom the housing 32 of the instrument A for replacement with anothermodule containing different beam splitter and filters associated withanother selected primary dye. The instrument includes a lamp subhousing33 and a camera subhousing 35.

In an example (FIG. 3), each module includes a mounting block 34 with aflange 36 that is affixable to the housing with a single screw 38. Thebeam splitter 6 is held at 45° in the block with a frame 40 and screws42. The emission filter 8 mounts (e.g. with glue) into the block. Theexcitation filter 7 mounts similarly into a mounting member 44 that isheld by screws 46 to the block. With the module in place, the instrumentis closed up with a side plate 47 that is screwed on. Positioning pins(not shown) ensure repeatable alignment. The replacement module may havethe same mounting block and associated components, with the beamsplitter and filters replaced.

The detector lens 9 (FIG. 1) is cooperative with the vial lenses 2 b andthe field lens 3 to focus the individual beams on the detector 10. Thelens should be large aperture, low distortion and minimum vignetting.

The detector preferably is an array detector, for example a chargeinjection device (CID) or, preferably, a charge coupled device (CCD). Aconventional video camera containing a CCD detector, the detector lensand associated electronics for the detector should be suitable, such asan Electrim model 1000L which has 751 active pixels horizontal and 242(non-interlaced) active pixels vertical. This camera includes a circuitboard that directly interfaces to a computer ISA bus. No framegrabbercircuitry is required with this camera. Essentially any other digitalimaging device or subsystem may be used or adapted that is capable oftaking still or freeze-frame images for post processing in a computer.

A detector with a multiplicity of photoreceptors (pixels) 78 ispreferable if there are a plurality of vials, to provide separatemonitoring of each. Alternatively a scanning device may be used with asingle photodetector, for example by scanning the folding mirror andusing a small aperture to the detector. Also, a simple device such as aphotomultipier may be used if there is only one vial. A CCD receiveslight for a selected integration period and, after analog/digitalconversion, reads out digital signal data at a level accumulated in thisperiod. The integration is effectively controlled by an electronicshutter, and a frame transfer circuit is desirable. Signal data aregenerated for each pixel, including those receiving the individual beamsof emitted light from the vials.

The instrument preferably includes a fluorescent reference member 4 thatemits reference light in response to the excitation beam. Advantageouslythe reference member is formed of a plurality of reference emitters,e.g. 6, each emitting a reference beam of different intensity inresponse to the excitation beam. The range of these intensities shouldapproximate the range of intensities expected from the marker dye in thevials; for example each segment may be separated in brightness by abouta factor of 2.5. The reference member is disposed to receive a portionof the excitation beam from the beam splitter. A good location isadjacent to the field lens, so that the optical paths associated withthe member approximate those of the vials. Most of the reference lightpasses back through the beam splitter as a reference beam to thedetector. The detector pixels receive the emission beam to generatereference signals for utilization along with the data signals in thecomputing of the concentration of DNA.

Advantageously the reference member 4 (FIG. 4) comprises a plasticfluorescent strip 4 a and a neutral density filter 4 b mounted over thefluorescent strip, optionally with an air space 4 h between, such that aportion of the excitation beam and the reference beam are attenuated bythe neutral density filter. The neutral density filter has a series ofdensities 4 c to effect the plurality of reference emitters (segments)each emitting a reference beam of different intensity. A heating strip 4d and an aluminum strip 4 g to smooth the heating are mounted in atrough 4 e on the bottom thereof, and the fluorescent strip is mountedon the aluminum strip over the heating strip. To prevent heat loss, thisassembly preferably is covered by a transparent plexiglass window (notshown, so as to display the varying density filter). To help maintainconstant fluorescence, the heating strip is controlled to maintain thefluorescent strip at a constant temperature against the thermal cyclesof the cycler block and other effects. This is done because mostfluorescent materials change in fluorescence inversely with temperature.

The computer processor 14 (FIG. 1) may be a conventional PC. Thecomputer programming is conventional such as with “C”. Adaptations ofthe programming for the present invention will be readily recognized andachieved by those skilled in the art. The processor selectivelyprocesses signals from pixels receiving light from the vials and thereference emitters, ignoring surrounding light. The programmingtherefore advantageously includes masking to define the pixel regions ofinterest (ROI), e.g. as disclosed in copending provisional patentapplication Ser. No. 60/092,785 filed Jul. 14, 1998 of the presentassignee. Mechanical alignment of the optics may be necessary tocooperatively focus the beams into the programmed regions of interest.The analog data signals are fed to the processor through ananalog/digital (A/D) device 15 which, for the present purpose, isconsidered to be part of the processor. A saturation level is proscribedby either the detector or the A/D or, preferably, the CCD dynamic rangeis matched to the A/D dynamic range. A suitable range is 8 bits ofprecision (256 levels), and the CCD amplifier offset is set so that thedark signal output of the CCD (with the shutter 12 closed) is within theA/D range. The processor instructs the detector with selected exposuretime to maintain the output within the dynamic range.

In a typical operation, fluorescence data are taken from the pluralityof vials (e.g. 96 regions of interest) and from the reference emittersegments, for each cycle in a DNA reaction replication sequence ofthermal cycles, typically 40 to 50. Two data sets are taken (FIG. 5) foreach thermal cycle during the extension phase of the PCR reaction atabout 60° C., which is the phase where all of the DNA strands haverecombined into double strands. One set is normal primary data 50 (alongwith reference data described below) and the other is dark signal data51 with the mechanical shutter closed. Both digital data sets 50, 51 areconverted by the A/D 15 from respective analog data signals 48, 49 fromthe detector. The dark are subtracted 55 from the normal, to yielddark-corrected data 57. In a simple procedure, the subtraction is pixelby pixel. Alternatively, total dark for each region of interest aresubtracted from corresponding total fluorescence data. In anotheralternative, in order to increase effective dynamic range, it isadvantageous to collect multiple exposures during each exposure period,e.g. 4 or 8 exposures. This is done by collecting multiple normalexposures and dark signal data for each pixel, subtracting eachrespective dark image from the normal data, then adding the subtracteddata together to yield the primary data. This improves the statisticalvalidity of the image data and increases its effective dynamic range.

Data are taken simultaneously from the reference strip which has, forexample, 6 segments together with the 96 vials for a total of 102regions of interest. Preferably the processing means provides forautomatic adjustment of the exposure time to maintain the data signalswithin a predetermined operating range that is less than the saturationlimit during the DNA replication sequence, for example 35% to 70% ofsaturation. Computations for DNA concentration include corrections inproportion to adjustments in exposure time (FIG. 6). Signal data 50, 51from each exposure 52, 53 are obtained during a previously determinedexposure time 54 by totaling the pixel counts within each region ofinterest (ROI).

To provide the time adjustments, the highest signal data 56, which isdata from one or more highest pixel readings, such as the threehighest-reading contiguous pixels, is searched out 58 from thecorresponding data signals 50. From a comparison 62 it is determinedwhether the highest signal data are less than, within or higher than theselected operating range 60. Based on such determination, the exposuretime is adjusted 64, i.e. increased, retained or reduced, to obtain thesubsequent exposure time 66. A reference time 68 (FIG. 5) also isselected which may be, for example, an initial time or a fixed standardtime such as 1024 ms. The dark-corrected data 57 is time-corrected 69 toyield corrected primary data 71, dividing by ratio of actual exposuretime to the reference time. The first several cycles may be out ofrange, and thereafter a useful fluorescence curve should be obtained(FIG. 7).

For the reference emitter, from the pixels receiving light from thereference strip 4 (FIGS. 1 and 4) reference data signals 73 aregenerated and converted by the A/D 15 to reference data 72. Selectedreference data 74 from a specific reference segment 4 c (FIG. 4) areselected 76 as that data having the highest signal strength that is lessthan a predetermined maximum 77 that, in turn, is less than thesaturation limit, e.g. 70%. A next dimmer segment is also selected 75,and the selected reference data 74 include the data from that segment.The dark data 51 are subtracted 78 from the reference data 74, and thedark-corrected data 80 are adjusted 84 for exposure time 54 to yieldadjusted reference data 82.

The data 82 includes dark corrected data 82′ for the highest segment anddark corrected data 82″ for the next dimmer segment (FIG. 9). The ratiosof brightness between each segment are computed 89 and built up over thecourse of data collection. Each time data is collected, the ratiobetween the highest and next dimmer segment is calculated. As differentoptimum segments are selected on succeeding data collections, a table ofratios 85 is assembled. Alternatively, these rations may be collectedand calculated in advance.

This adjusted reference data 82′ (from data 82, FIG. 5) are utilized forcomputing normalized reference data 88 which are normalized 86 in realtime as a ratio to reference data 90 from an initial or other selectedprevious cycle in the DNA replication (PCR) sequence by working backwith the ratios 85. The normalized reference data are utilized on thecorrected primary data 71 in a normalization computation 92 to providedrift normalized primary data 94 by dividing the primary data by thenormalized reference data. This corrects for instrument drift during themonitoring. DNA concentration 96 may then be computed 98 from a storedcalibration factors 99, determined by running standard known DNAconcentrations to determine the slope and intercept of a line relatingstarting concentration to the starting cycle of the growth curve (FIG.7) as taught in the aforementioned article by Higuchi and U.S. Pat. No.5,766,889. (Further normalization 118, 120 and baseline correction122-130 are discussed below.)

Extension phase data for a typical PCR sequence would look like FIG. 7,plotted for each PCR cycle. If desired, the data may be corrected fordye bleaching or other sample chemical effects by normalizing to samplevials containing samples with the same dye and with DNA amplificationprevented chemically.

The sample additionally may contain one or more types of dye moleculesthat serve as a “passive” reference having some fluorescence in the samewavelength range as the DNA binding dye. This reference dye is made up,for example, of a nucleic acid sequence labeled with Rhodamine andFluorescein dye derivatives. A suitable reference is Rox dye fromPerkin-Elmer Applied Biosystems. These passive dye molecules do not takepart in the PCR reaction, so that their fluorescence is substantiallywithout influence from DNA and remains constant during the reaction.This fluorescence can be used to normalize the fluorescence from the DNAbinding dye with a standard concentration of passive dye included in theingredients of at least one vial, preferably in every vial.

The source beam includes a secondary excitation frequency that causesthe passive dye to fluoresce at a secondary frequency and thereby emit asecondary beam directed to the detector to generate correspondingsecondary data signals. The processor is receptive of the secondary datasignals for computing secondary data representative of standardconcentration. These data are used to normalize the primary data, sothat the concentration of DNA is normalized to the standardconcentration of passive dye after correcting computations ofconcentration of DNA in proportion to adjustments in exposure time, andin conjunction with the normalization for drift. Advantageously, and inthe present example, the secondary excitation frequency is identical tothe primary excitation frequency, and the passive dye fluoresces suchthat the emitted secondary beam is substantially at the emissionfrequency. The primary data signals are generated during each extensionphase of cycling of the thermal cycler block when DNA is recombined andcorrespondingly primary dye emission is maximized. The secondary datasignals are generated during each denature phase of cycling of thethermal cycler block when DNA is denatured and correspondingly primarydye emission is minimized. Thus data signals for the primary phase aresubstantially representative of DNA concentration, and data signals forthe secondary phase are substantially representative of the standardconcentration of passive dye.

The dark and normal data are taken for the vial samples and thereference strip, and the dark is subtracted from the normal fluorescencedata. This dark and normal data set is taken during the extension phaseof the PCR reaction at about 60° C., which is the phase where all of theDNA strands have recombined into double strands. During this phase, thefluorescence from the DNA binding dye is maximized, and the fluorescencefrom the passive reference molecules is superimposed but much smaller. Aseparate dark and normal data set is taken during the high temperature(about 95° C.) denaturing phase, during which the DNA is denatured orsplit into single strands. During this phase, the fluorescence of theDNA binding dye is minimized, and almost non-existent, because the DNAis not double stranded and the fluorescence of the dyes used have alarge decrease in fluorescence with increased temperature. Therefore thedenaturing phase images substantially contain reference fluorescencefrom the passive reference molecules. The dark-corrected reference(denaturing) data set, after correction for measured temperaturedependence, may be subtracted from the dark-corrected DNA binding dyedata set, or may be deemed insignificant for the normal data set.

Alternatively, it may be desirable to image the passive reference dyelabeled molecules by taking the additional images, for each PCR cycle,using a separate optical bandpass filter that rejects wavelengthsemitted by the DNA binding dye while accepting wavelengths from thepassive reference dye. This data would be functionally equivalent to thedenature data.

Illustrating operation for the denature phase (FIG. 8), respectivenormal and dark data signals 102, 104 are obtained in the same manner asfor the primary data, with normal exposure 52′ and closed shutter 53′.Exposure time 106 may be the same as for an adjacent extension phase inthe sequence, or determined from a previous denature phase run (asdescribed with respect to FIG. 7), or may be a suitable timepredetermined for all denature phases in the sequence. The A/D 15converts the signals to secondary data 108 and dark data 110. The darkis subtracted 55′ from the secondary to yield dark-corrected data 112which is further corrected 69′ with a reference time 114 and the actualexposure time 106 to yield corrected secondary data 116.

The extension cycle, drift normalized primary data 94 then arenormalized 118 by dividing by the average of a selected number (e.g. 10)of cycles for the denature phase corrected secondary data 116 to producefurther normalized fluorescence data or further normalized data 120,which removes sample well to well non-uniformity effects. Cycle by cycledivision may be used in place of an average. Alternatively the secondarydata may be applied to the corrected primary data 71 before or afterdrift normalization. Baseline samples may be selected 122 and averaged124 to produce baseline data 126. The further normalized data 120 arethen divided 128 by the baseline data to yield baseline corrected data130. These baseline samples are selected so as to be before the PCRgrowth exceeds the nearly horizontal base line portion of the curve inFIG. 7. Selected baseline cycles may be, for example, cycles 6 through15. After further normalization 118, the further normalized data 118 areused to compute 98 DNA concentration 96.

The trend (e.g. least squares regression line) of these same baselinesamples is subtracted from the normalized extension cycle data, toproduce data that has a flat base line at zero. This data set may thenbe processed using established or other desired PCR methods to calculatethe amount of starting copies of DNA. A simple procedure is toextrapolate for the inflection point at the transition from flat torising. A more sophisticated procedure is described in theaforementioned U.S. Pat. No. 5,766,889.

The data may be used for various purposes, for example quantitativemonitoring of the reaction or determination of replicated DNAconcentration, or determination of the to starting amount. Theinstrument also may be used (with or without normalizations and othercorrections) simply to display whether replication is taking placeduring a sequence, or has taken place.

While the invention has been described above in detail with reference tospecific embodiments, various changes and modifications which fallwithin the spirit of the invention and scope of the appended claims willbecome apparent to those skilled in this art. Therefore, the inventionis intended only to be limited by the appended claims or theirequivalents.

1-17. (canceled)
 18. An instrument comprising: an apparatus configuredto hold a plurality of spaced-apart reaction regions; a light sourceconfigured to direct an excitation beam toward the apparatus; a detectorconfigured to receive emission beams from the apparatus; an excitationbeam path disposed from the light source to the apparatus; an emissionbeam path disposed from the apparatus to the detector; and a lensdisposed along the excitation beam path and along the emission beampath, the lens configured during use to direct the excitation beam tomore than one of the reaction regions simultaneously.
 19. The instrumentof claim 18, wherein the light source comprises a laser.
 20. Theinstrument of claim 18, further comprising a dichroic reflectorconfigured to reflect the excitation beam.
 21. The instrument of claim18, wherein the light source is configured to emit at least twoexcitation beams, each excitation beam characterized by a centralwavelength that is different from that of the other excitation beam(s).22. The instrument of claim 21, further comprising a plurality ofdichroic reflectors configured to reflect the at least two excitationbeams from the light source.
 23. The instrument of claim 18, whereinlens comprises a focusing optic.
 24. The instrument of claim 18, whereinthe lens is located proximal to the apparatus.
 25. The instrument ofclaim 18, wherein the instrument comprises the plurality of spaced-apartreaction regions and at least some of the reaction regions comprise oneor more of a component for DNA replication, a fluorescent dye, or a dyelabeled probe.
 26. The instrument of claim 18, wherein the instrumentcomprises the plurality of spaced-apart reaction regions wherein atleast two reaction regions each produce an emission beams thatsimultaneously passes through the lens and onto the detector.
 27. Theinstrument of claim 18, wherein the apparatus comprises a PCR reactionapparatus.
 28. The instrument of claim 18, wherein the apparatuscomprises a heater to maintain a temperature.
 29. An instrumentcomprising: an apparatus having a surface configured to hold a pluralityof spaced-apart reaction regions; a light source configured to generatean excitation beam; a detector configured to detect emission beams fromthe apparatus; an excitation beam path disposed from the light source tothe surface of the apparatus; an emission beam path disposed from thesurface of the apparatus to the detector; and a lens disposed along theexcitation beam path and the emission beam path, the lens configured todirect the excitation beam to the surface configured to hold a pluralityof spaced-apart reaction regions; wherein said lens is configured tosimultaneously illuminate a plurality of spaced-apart reaction regions.30. The instrument of claim 29, wherein the light source comprises alaser.
 31. The instrument of claim 29, further comprising a dichroicreflector configured to reflect the excitation beam.
 32. The instrumentof claim 29, wherein the light source emits at least two excitationbeams, each excitation beam characterized by a central wavelength thatis different from that of the other excitation beam(s).
 33. Theinstrument of claim 32, further comprising a plurality of dichroicreflectors configured to reflect light from the light source.
 34. Theinstrument of claim 29, wherein lens comprises a focusing optic.
 35. Theinstrument of claim 29, wherein the apparatus comprises a PCR reactionapparatus.
 36. The instrument of claim 29, wherein the apparatuscomprises a heater to maintain a temperature.
 37. An instrumentcomprising: an apparatus having a portion configured to hold a pluralityof spaced-apart reaction regions; a light source configured to direct anexcitation beam toward the portion; a detector configured to receiveemission beams from the portion; and a lens configured during use todirect the excitation beam to the portion, to illuminate more than onereaction region simultaneously, and to direct emission beams from theportion to the detector to produce a data signal.
 38. The instrument ofclaim 37, wherein the light source comprises a laser.
 39. The instrumentof claim 37, further comprising a dichroic reflector configured toreflect the excitation beam.
 40. The instrument of claim 37, wherein thelight source emits at least two excitation beams, each excitation beamcharacterized by a central wavelength that is different from that of theother excitation beam(s).
 41. The instrument of claim 40, furthercomprising a plurality of dichroic reflectors configured to reflectlight from the light source.
 42. The instrument of claim 37, whereinlens comprises a focusing optic.
 43. The instrument of claim 37, whereinthe apparatus comprises a PCR reaction apparatus.
 44. The instrument ofclaim 37, wherein the apparatus comprises a heater to maintain atemperature.